Abstract
Engagement of the receptor CD244 (2B4) by its ligand CD48 has inhibitory and activating potential, and this differs depending on experimental systems in mouse and human. We show that, in both mouse and human upon engagement of its ligand CD48, CD244 can give a negative signal to natural killer cells, implying conservation of function between the two species. The signaling mechanisms used by CD244 in both human and mouse are conserved as shown by quantitative analyses of the direct molecular interactions of the SH2 domains of the adaptors SLAM-associated protein (SAP) and EAT-2 and of FYN kinase with CD244 together with the indirect interactions of the FYN SH2 domain with EAT-2. Functional experiments support the biochemical hierarchy of interactions and show that EAT-2 is not inhibitory per se. The data are consistent with a model in which the mechanism of signal transduction by CD244 is to regulate FYN kinase recruitment and/or activity and the outcome of CD48/CD244 interactions is determined by which other receptors are engaged.
Highlights
Signaling by receptors that bind the intracellular adaptor SAP2 is important for immune regulation in combating infection, and it is dysregulated in cancer and autoimmunity [1, 4]; an understanding of the mechanisms involved has wide implications
Of three monoclonal antibodies (mAbs) that were specific for mouse CD244 and bound to a mouse T cell hybridoma transduced with mouse CD244 (Fig. 1b), one completely blocked binding of CD48CD4d3ϩ4-coated fluorescent beads to cells [30] expressing CD244 (Fig. 1c). mAb OX121 partially blocked binding of CD48-CD4d3ϩ4-coated beads, similar to a mAb used in our previous study [11]
Dissection of the pattern of binding by intracellular proteins to the tyrosine motifs in the cytoplasmic regions of mouse and human CD244 showed that intracellular interactions are conserved
Summary
Recombinant Proteins—The rat basophil leukemia cell line was stably transfected with the pBabe vector encoding a chimeric receptor containing the extracellular region of the C57/ BL6 allele of mouse CD244 [11] and the transmembrane and cytoplasmic regions of the mouse -chain, CD244- [28], using FuGENETM transfection reagent. Concentration was determined using absorbance at 280 nm and the following theoretical extinction coefficients (Vector NTI): mouse SAP and SH2, 23,140 and 23,020 MϪ1 cmϪ1; human SAP and SH2, 24,660 and 24,540 MϪ1 cmϪ1; mouse EAT-2, 19,090 MϪ1 cmϪ1; human EAT-2 and SH2, 21,980 and 15,130 MϪ1 cmϪ1; human FYN SH2, 26,510 MϪ1 cmϪ1; human FYN SH3, 29,280 MϪ1 cmϪ1; mouse FYN SH3-SH2, 35,680 MϪ1 cmϪ1; and human FYN SH3-SH2, 37,561 MϪ1 cmϪ1 For mammalian expression as enhanced green fluorescence protein (EGFP) fusion proteins, BamHI-HindIII fragments from pTrcHISA constructs were cloned into the retroviral vector pLEGFP-C1 (Invitrogen) and expressed in 2B4 hybridoma cells as described for the pBabe vector, and selection was maintained with G418 (0.4 mg/ml).
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